In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and other connective tissue symptoms and then apply the knowledge gained from our studies to the treatment of children with these conditions. Our Branch has generated a knock-in murine model for OI with a classical collagen mutation. Using Brtl, we completed a major theraeputic trial of bisphosphonate, which complements our pediatric trial. We compared femora from treated and untreated Brtl and wild-type littermates. On the one hand, alendronate treatment increased femoral DXA and cortical volumetric BMD, TbN and cortical thickness, as well as load to fracture. However, detrimental changes were detected in material and cellular parameters of bone, affecting material strength and brittleness. Furthermore, dramatic retention of mineralized cartilage disrupts matrix continuity and may contribute to the weakening of bone material. In addition, bone cells function declined and osteoblasts were altered to a flattened morphology, similar to lining cells. These studies contribute to the increased cautionary notes in the literature concerning avoidance of an elevated cummulative bisphosphonate dose. Brtl is also being used as the model for testing an anabolic therapy for OI anti-sclerostin antibody which works by stimulating bone fomation along the canonical wnt pathway. When growing young Brtl mice were treated with Scl-Ab for 5 weeks, Brtl femora increased cortical bone formation, which improved mechanical strength to WT levels, without further exacerbating the underlying brittleness of OI bone material. Trabecular bone formation was also stimulated in Brtl mice by SclAb treatment, although to a lesser extent that in WT mice. In contrast, administration of Scl-Ab to adult 6 month old Brtl mice resulted in improved trabecular and cortical bone mass and serum osteocalcin, leading to improved bone stiffness and mechanical strength. These data present the encouraging prospect of the potential for new bone formation in adults with OI. Brtl is currently being used for trials of two non-traditional therapies. In a collaborative study, in utero cell transplantation of stem cells expressing GFP showed improved femoral geometry and biomechanics, despite a low level of engraftment. These results are encouraging for translational trials. Second, we are modelling a lesson from type I OI. Specific suppression of transcripts from the mutant collagen allele can biochemically transform individuals with severe OI into mild type I OI. The RZ target site in the Brtl mutant allele is being targeted by matings with transgenic mice expressing ribozymes targeted to the Brtl mutation. In complementary suppression studies, allele specific siRNA was shown to reduce mutant collagen expression by 40%. We have identified a novel high bone density form of OI caused by mutation in the C-proteinase cleavage site. The Asp-Ala dipeptide between he telopeptide and the C-propeptide of each chain is cleaved by C-proteinase/BMP1 to release mature collagen. Children with substitutions at these residues present with fractures and a high DEXA z-score. Interestingly, despite the high DEXA, radiographs and histomorphometry are similar to type I OI and point to matrix deficiency. Pericellular processing of procollagen C-propeptide is delayed. FTIR and BSEM are being used to study the amount and crystallinity of bone samples from our two probands. These data not only reveal a novel form of OI but also provide new fundamental information on roles of procollagen processing and the mechanism of tissue mineralization. To better understand the relationship of genotype and phenotype in human OI, the BEMB led and international consortium of connective tissue laboratories to assemble and analyze a mutation database containing over 830 mutations. Genotype-phenotype modeling revealed different functional relationships for each chain of type I collagen. Lethal mutations in alpha 1 (I) coincide with the Major Ligand Binding Regions. Lethal regions in alpha 2(I) continue to support the Regional Model first proposed by the BEMB, with lethal mutations in regularly-spaced clusters along the chain that coincide with proteoglycan binding regions. This model correctly predicts clinical outcome in 86% of alpha 2(I) mutations. The consortium database now contains over 1300 mutations and the genotype-phenotype analysis is being updated. In bench studies aimed at understanding the basis of the phenotypic variability of patients with the identical OI-causing mutation, we collaborated on investigations of cellular cytoskeleton in Brtl lethal and surviving mice. Components of intermediate filaments, microtubules and actin fibaments were all shown to be abnormal only in tissues from lethal mice. The aberrant cytoskeleton affects TGF-b and integrin signaling. This data was extended to cells from patients with lethal and non-lethal mutations caused by identiacal glycine substitutions. They point to the cytoskeleton as a phenotypic modulator and potential novel target for OI treatment. We are also continuing our clinical studies of children with types III and IV OI. The BEMB undertook the first randomized controlled trial of bisphosphonate in children with types III and IV OI. The aim was to test both the primary skeletal gains and secondary gains (improved functional level and muscle strength and decreased pain) reported in observational trials. The treatment group experienced improvement in vertebral parameters, including BMD z-scores, central vertebral height and vertebral area. However, the increment in vertebral BMD in the treatment group tapered off after one to two years of treatment.There was no significant change in ambulation level, lower-extremity strength or pain in children with OI treated with pamidronate. Hence the changes previously reported appear to have been a placebo effect in uncontrolled trials. We are recommending that treatment of children with types III and IV OI with pamidronate be limited to at most three years, with subsequent follow-up of bone status. Furthermore, we are currently engaged in a dose comparison trial. We are also focusing on the variability of response to treatment in each group. The improvements in vertebral height and area do not correlate with changes in DXA z-score, nor did the improvement in vertebral height and area correlate for individual children. These differences may be related to important individual variation in ability to synthesize new bone or to remodel bone. They also highlight the inadequacy of DXA as a surrogate for bone strength.